Heat shock protein 72/73 (Hsp70) is a cytosolic molecular chaperone that carrys out fundamental roles under both normal and stress situations. There is great interest in delineating the mechanisms whereby Hsp70 levels are regulated. Here it is demonstrated that N-acetyl-leucyl-leucyl-norleucinal (ALLN), a synthetic tripeptide which inhibits cysteine proteases and inhibits proteasomes, can markedly induce Hsp70 levels (up to 30-fold above baseline in HepG2 cells and human endothelial cells). Induction of Hsp70 by ALLN is dose-dependent and not related to cell toxicity. ALLN selectively increases Hsp70 levels without affecting Hsp25, Hsp27, Hsp60, Hsp86, Hsp90, Hsp104 or immunoglobulin heavy chain binding protein (Bip) in HepG2 cells. A series of other protease inhibitors that were examined had no effect on Hsp70, except for N-acetyl-leucyl-leucyl-methioninal (ALLM), which is highly similar to ALLN both in its structure and protease inhibitory features. ALLN induces Hsp70 not only by stabilizing the protein but also by dramatically increasing its synthesis. The modulation of Hsp70 synthesis by ALLN appears to result from a rapid and marked increase in transcription of the hsp70, i.e., hsp72 gene, since the induction of hsp70, i.e. hsp72, mRNA was blocked in cells co-treated with actinomycin D. hsp70 (i.e. hsp72) mRNA levels are affected by the duration of exposure to ALLN: significant elevations occur within 60 min. of treatment, and a decline to background levels is observed by 7 hours of recovery. The ALLN-induced increase in hsp70 (i.e. hsp72) gene expression is associated with trimerization of the heat shock transcriptional factor (HSF1). ALLN does not affect the steady-state HSF1 protein level. The effects of ALLN appear to require de novo protein synthesis, since the induction of both HSF1 trimerization and hsp70 (i.e. hsp72) transcription is blocked by co-treatment with cycloheximide. These results suggest that a cysteine protease may be involved in the regulation of Hsp70 synthesis via effects on the hsp70 transcriptional factor, HSF1. This cysteine protease may normally degrade a rapidly turning-over protein involved in the trimerization of HSF. Of a series of protease inhibitors that were tested, only the related aldehydic tripeptides, N-acetyl-leucyl-leucyl-methioninal (ALLM) and the proteasome inhibitor, Cbz-leucyl-leucyl-leucinal (MG132) induced Hsp70 levels. The specific proteasome inhibitor, lactacystin, which has a different structure, also induced Hsp70 levels. Overall, these results suggest that a rapidly turning-over protein which is normally degraded by proteasomes may be involved in the regulation of Hsp70 synthesis via effects on the hsp70 transcriptional factor, HSF1.
Induction of heat shock (stress) proteins (Hsps) , a class of molecular chaperones is a physiological and biochemical response to an abrupt increase in temperature (Ashburner, et al., 1979; Lindquist, 1986) and to a variety of other metabolic insults (Craig, 1985; Morimoto, et al., 1994), including exposure to heavy metals, amino acid analogs, toxins and oxidative stress. This response is found in all prokaryotic and eukaryotic cells and is characterized by a repression of normal protein synthesis together with the rapid initiation of transcription of several Hsp-encoding genes (Lindquist, 1986). Among these highly conserved Hsp family members are two nearly identical, cytosolically located heat shock proteins, Hsp72 (the inducible form) and Hsp73 (the constitutively synthesized form). These two proteins, commonly referred to as cytosolic Hsp70, function as molecular chaperones and play fundamental roles in a number of important biological processes. Under nonstressed conditions, Hsp70 interacts transiently with nascent polypeptides to facilitate proper folding and maturation and to promote protein translocation across mitochondrial and endoplasmic reticulum (ER) membranes (Hartl and Martin, 1992; Deshaies, et al., 1988; Dierks, et al., 1993; Neupert and Pfanner, 1993). During stress conditions, Hsp70 forms a complex with proteins that misfold or unfold, thus either "rescuing" these proteins from irreversible damage or degradation (Hightower, 1991; Gaitanaris, et al., 1991; Gething and Sambrook, 1992; Craig, et al., 1994) or increasing their susceptibility to proteolytic attack (Hayes and Dice, 1996).
Recently, elevated expression of Hsp70 and other Hsps has been observed in cells and tissues under conditions representative of human diseases, including ischemia, oxidant injury, atherosclerosis and aging (Marber, et al., 1988; Minowada and Welch, 1995; Johnson, et al., 1995). The increased expression of these stress proteins could represent an acute response to altered physiological states, as well as chronic adaption to particular diseases. The primary function of these stress responses is thought to be cytoprotective. For example, overexpression of Hsp70 alone was demonstrated to protect cells from thermal injury and to increase cell survival (Angelidis, et al., 1991; Li, et al., 1991). Elevated levels of inducible Hsp70 have been associated with improved post-ischemic recovery (Currie, et al., 1988) and tolerance to ischemia in gerbil hippocampal neurons (Ohtsuki, et al., 1992). It has also been reported that both heat shock-induced and exogeneous Hsp70 can protect smooth muscle cells from serum deprivation-induced cell death (Johnson, et al., 1995). Overexpression of Hsp70 also protects murine fibroblasts from both ultraviolet (UV)-light injury and proinflammatory cytokines released during UV-exposure (Simon, et al., 1995). The protective role of Hsp70 was demonstrated clearly by two recent studies with transgenic mice in which overexpression of human inducible Hsp70 protected myocardium from ischemic reperfusion injury (Marber, et al., 1995; Plumier, et al., 1995) and enhanced postischemic recovery of the intact heart (Rodford, et al., 1996). These potential clinical applications of Hsp70 have stimulated investigators to search for efficient pharmacological means of rapidly and selectively inducing Hsp70.
Studies of the involvement of molecular chaperones in the assembly and secretion of apolipoprotein B100 (apoB)-containing lipoprotein from cultured liver (HepG2) cells have been performed. ApoB is a very large, extremely hydrophobic secretory protein that appears to be constitutively translated but inefficiently translocated across the ER membranes (Dixon and Ginsberg, 1993; Ginsberg, 1995). As a result, nascent apoB assumes a transmembrane topology with some portion of the nascent protein exposed to the cytosol. Since the extreme hydrophobicity of apoB makes it unlikely that it would maintain a translocation-competent conformation in the cytosol without the "assistance" of a chaperone, a possible association of apoB with Hsp70 was investigated (Zhou, et al., 1995). It was found that Hsp70 associated transiently with nascent apob and that this interaction appeared to be regulated by the translocation status of apoB. Less apoB was bound to Hsp70 in the presence of oleic acid, which facilitates apoB translocation across the ER membranes by stimulating new triglyceride synthesis. In contrast, more apoB was bound to Hsp70 in the presence of a cysteine protease inhibitor, N-acetylleucyl-leucyl-norleucinal, (ALLN), which is also a proteasome inhibitor (Rock, et al., 1994; and Ward, et al., 1995), which protects apoB from degradation without enhancing translocation. During these studies, a marked, unexpected increase in Hsp70 levels in cells treated with ALLN was observed. The studies presented here were designed to determine the mechanisms underlying the induction of Hsp70 by ALLN.